In addition, we observed that the PKC activator, PMA, as well as a bacterial fermentation end product, butyrate, also regulated TSLP expression both at the mRNA and protein level. Moreover, a strong synergistic effect between PMA and butyrate

was observed. The latter effect may be physiologically relevant given the major biological function of butyrate as an energy source in the colon [30] as well as its function as an epigenetic regulator [31]. As expected, stimulation of IECs by IL-1 induced NF-κB translocation into the nucleus and TSLP transcription involving IKK-β activity as revealed by the specific inhibition induced by Bay 11–7082. Clearly, the functional importance of both p38 and PKA was also identified using SB203580 and H-89, respectively. Conversely, extracellular signal-regulated kinase (ERK) ABT-888 in vivo had little effect since UO126 barely inhibited TSLP transcription. We first postulated that both p38 and PKA may act independently of IKK-β involvement since their specific pathway inhibitors were effective in the presence of Bay 11–7082, whereas UO126 had no effect. However, when transient transfections were performed with a 4 kb TSLP-promoter region, mutated for NF2 binding site, the stimulatory effect of IL-1 was completely abolished;

thus Peptide 17 in vivo arguing for a NF-κB only dependent regulation. We present in Figure 7 our working hypothesis that can explain the overall results obtained in IL-1-dependent TSLP regulation. Considering that, in the presence of BAY 11–7082, the effects of both p38 and PKA inhibitors are still apparent, we can argue that, since BAY 11–7082 has an IC50 of 10 μM [32], at the concentration 20 μM used Fossariinae in the current study, IKK-β may only be partly inhibited and that the remaining TSLP transcription activity is still mediated by IKK-β. This has been verified using a NF-κB-dependent SEAP reporter system [33]. In fact, at the 20 μM concentration, BAY 11–7082 only inhibited IL-1-dependent NF-κB activation

by about 60% in Caco-2 cells. To explain the effects of the p38 inhibitors, our hypothesis is that IL-1 is activating IKK-β by two separate modes; first via the classical IL-1 receptor associated kinase/TGF-β activated kinase (IRAK/TAK) dependent pathway and second via a MKK/p38-dependent pathway as revealed previously for IL-6 [34]. Thus, inhibition of p38 resulted in a decreased TSLP expression due to a reduced activation of IKK-β, and enhances BAY 11–7082 direct inhibitory activity. Considering the involvement of PKA, it has been shown that PKA can also interfere with the NF-κB pathway; indeed PKA was revealed to phosphorylate p65 in a cAMP-independent manner therefore increasing transcriptional activity [35]. Our results argue for a similar regulation of TSLP transcription in human IECs. Recently, TSLP has been shown to be regulated by NF-κB in both human and mice airway epithelial cells [16]. A site located at –3.